Project summary/abstract Arterial calcification has increasingly been associated with poor outcomes in a wide variety of patient populations including those with coronary, aortic, and peripheral artery disease, as well as patients with diabetes, and renal failure. We previously demonstrated that calcification in peripheral arteries predicts major amputation even after adjusting for demographics, cardiovascular risk factors, and the ankle brachial index. During arterial calcification, medial smooth muscle cells (SMCs) undergo osteogenic transformation with loss of phenotype-specific markers and new expression of factors and signaling molecules most commonly found in developing bone. We and others have established a critical role for matrix metalloproteinases (MMPs) in the progression of arterial calcification. MMPs are highly expressed in calcifying arteries, and we have demonstrated that reducing their activity with broad spectrum and synthetic inhibitors can prevent experimental calcification both in vitro and in vivo. In preliminary data for the current proposal, we show that MMP-3 (stromelysin-1) is the most strongly induced of all metalloproteinases in calcifying rodent aortas. Reducing MMP-3 activity decreases calcium accumulation in cultured human and rodent SMCs and in a rat aorta organ culture model. Aortas from MMP-3-deficient mice are protected from medial calcification in organ culture and in vivo. The location of MMP-3 activity required for calcification, whether local or systemic, and its contribution to SMC transformation is not currently known and this may act as a barrier to development of appropriate inhibitors for use in clinical trials. MMPs are primarily known for their ability to degrade extracellular matrix, yet recent evidence from a variety of cell types suggests an intracellular role for MMP-3 in phenotypic changes. Based on our data and those of others, we propose the overarching hypothesis that MMP-3, induced by inflammation and elevated phosphate levels, promotes osteogenic transformation of vascular SMCs and medial calcification through both local and systemic effects. We will begin to test this hypothesis by first defining the role of MMP-3 in phosphate-induced osteogenic transformation of vascular SMCs. We will next determine whether MMP-3 promotes medial calcification through systemic effects on circulating factors or through local actions within the arterial wall. Finally, we will evaluate whether doxycycline, an antibiotic with MMP inhibiting properties can slow the progression of arterial calcification in a clinical trial. As we progress through these aims we will answer critical questions about the role of MMPs in arterial calcification and the potential for a dedicated clinical trial to assess the role of MMP inhibition strategies to reduce calcification and improve outcomes in our patients with vascular disease.